Transgenic animals and methods of use

ABSTRACT

The present invention comprises non-human vertebrate cells and non-human mammals having a genome comprising an introduced partially human immunoglobulin region, said introduced region comprising human V H  coding sequences and non-coding V H  sequences based on the endogenous genome of the non-human mammal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/367,809, filed Jul. 26, 2010 and is incorporatedherein by reference.

FIELD OF THE INVENTION

This invention related to transgenic vertebrates, and more specificallyto transgenic vertebrates for the development of human therapeutics.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

The genes encoding human and mouse immunoglobulins have been extensivelycharacterized. Berman et al. (1988) EMBO J. 7:727-738 describe the humanIg V_(H) locus comprising the V_(H,D) and J_(H) gene segments. Sakano etal. (1981) Nature 290:562-565 describe a diversity segment of theimmunoglobulin heavy chain genes. Blankenstein and Kruwinkel (1987) Eur.J. Immunol. 17:1351-1357 describe the mouse variable heavy chain region.The generation of transgenic animals, such as mice having variedimmunoglobulin loci, has allowed the use of such transgenic animals invarious research and development applications, e.g., in drug discoveryand basic research into various biological systems. The generation oftransgenic mice bearing human immunoglobulin genes is described inInternational Application WO 90/10077 and WO 90/04036. WO 90/04036describes a transgenic mouse with an integrated human immunoglobulin“mini” locus. WO 90/10077 describes a vector containing theimmunoglobulin dominant control region for use in generating transgenicanimals.

Numerous methods have been developed for replacing endogenous mouseimmunoglobulin regions with human immunoglobulin sequences to createpartially- or fully-human antibodies for drug discovery purposes.Examples of such mice include those described in, for example, U.S. Pat.Nos. 7,145,056; 7,064,244; 7,041,871; 6,673,986; 6,596,541; 6,570,061;6,162,963; 6,130,364; 6,091,001; 6,023,010; 5,593,598; 5,877,397;5,874,299; 5,814,318; 5,789,650; 5,661,016; 5,612,205; and 5,591,669.Many of the fully humanized immunoglobulin mice have antibody productionbelow normal rates due to less efficient V(D)J recombination, andlimited antibody production caused from partial gene complement. Othersin which the mouse coding sequence have been “swapped” with humansequences are very time consuming and expensive to create due to theapproach of replacing individual mouse exons with the syntenic humancounterpart.

Based on the foregoing, it is clear that a need exists for efficient andcost-effective methods of efficiently producing human antibodies. Moreparticularly, there is a need in the art for non-human vertebratescomprising human immunoglobulin regions and transgenic animals havingthe ability to properly respond to an antigen.

In accordance with the foregoing object transgenic nonhuman animals areprovided which are capable of producing an antibody with human Vregions.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The present invention comprises non-human vertebrate cells and non-humanvertebrates having a genome comprising an introduced partially humanimmunoglobulin region, said introduced region comprising humanimmunoglobulin variable region locus coding sequences and non-codingsequences based on the endogenous immunoglobulin variable region locusof the non-human vertebrate. Preferably, the transgenic cells andanimals of the invention have genomes in which part or all of theendogenous immunoglobulin region is removed.

At a minimum, the production of human monoclonal antibodies in non-humanvertebrates requires that the host have at least one locus that willexpress human heavy chain immunoglobulin proteins and one locus thatwill express human light chain immunoglobulin proteins.

In some aspects, the partially human immunoglobulin variable regionlocus comprises human V_(H) coding sequences and non-coding V_(H)sequences based on the endogenous V_(H) region of the non-humanvertebrate. In these aspects, the

partially human immunoglobulin variable region locus further compriseshuman D and J gene coding sequences and non-coding D and J genesequences based on the endogenous genome of the non-human vertebratehost.

In other aspects, the immunoglobulin region comprises an introducedregion comprising human V_(L) coding sequences and non-coding V_(L)sequences based on the endogenous V_(L) region of the non-humanvertebrate. More preferably, the introduced partially humanimmunoglobulin region comprising human V_(L) coding sequences furthercomprises human J gene coding sequences and non-coding J gene sequencesbased on the endogenous genome of the non-human vertebrate host.

In certain aspects, the vertebrate is a mammal, and preferably themammal is a rodent, e.g., a mouse or rat. In other aspects, thevertebrate is avian, e.g., a chicken.

In one specific aspect, the invention provides a method for generating anon-human vertebrate cell comprising a partially human immunoglobulinregion, said method comprising: a) introducing two or more recombinasetargeting sites into a non-human vertebrate cell and integrating atleast one site in the cell's genome upstream and at least one sitedownstream of a genomic region comprising an endogenous immunoglobulinvariable region locus; and b) introducing a partially humanimmunoglobulin variable region locus comprising human immunoglobulinvariable region coding sequences and non-coding sequences based on theendogenous immunoglobulin variable region of the non-human vertebratehost into the non-human vertebrate host cell via recombinase mediatedexchange.

In a specific aspect of this method, the introduced partially humanimmunoglobulin region comprises human V_(H) gene coding regions, andfurther comprises i) human D and J gene coding sequences and ii)non-coding D and J gene and pre-DJ sequences based on the endogenousgenome of the non-human vertebrate host. The partially humanimmunoglobulin regions are preferably introduced into the host cellusing recombinase targeting sites that are introduced upstream of theendogenous V_(H) immunoglobulin genes and downstream of the endogenous Dand J gene region.

In other aspects, the V_(H) gene coding regions derive (at leastpartially) from other sources—e.g., they could be rationally orotherwise designed sequences, sequences that are a combination of humanand other designed sequences, or sequences from other species, such asnonhuman primates.

In yet another specific aspect, the introduced partially humanimmunoglobulin region comprises human V_(L) gene coding regions, andfurther comprises i) human J gene coding sequences and ii) non-coding Jgene sequences based on the endogenous genome of the non-humanvertebrate host. The partially human immunoglobulin regions arepreferably introduced into the host cell using recombinase targetingsites that are introduced upstream of the endogenous V_(L)immunoglobulin genes and downstream of the endogenous J region.

Preferably, the partially human immunoglobulin region is synthesized asa single nucleic acid, and introduced into the non-human vertebrate hostcell as a single nucleic acid region. The partially human immunoglobulinregion may also be synthesized in two or more contiguous segments, andintroduced to the vertebrate host cell in these discrete segments. Thepartially human nucleic acid can also be produced using recombinantmethods and isolated prior to introduction of the nucleic acid to thenon-human vertebrate host cell.

In another preferred aspect, the method further provides deleting thegenomic region flanked by the two introduced recombinase sites prior tostep b).

In another aspect, the invention provides methods for generating anon-human vertebrate cell comprising a partially human immunoglobulinregion, said method comprising: a) introducing two or more site-specificrecombination sites that are not capable of recombining with one anotherinto the genome of a cell of a non-human vertebrate host, wherein atleast one recombination site is introduced upstream of an endogenousimmunoglobulin variable region locus and at least one recombination siteis introduced downstream of the endogenous immunoglobulin variableregion locus; b) providing a vector comprising a partially humanimmunoglobulin region having i) human immunoglobulin variable regioncoding sequences and ii) non-coding sequences based on an endogenousimmunoglobulin variable region to the host cell, wherein the partiallyhuman region is flanked by the same two site-specific recombinationsites that flank the endogenous variable immunoglobulin region of thehost cell of a); c) introducing the vector of step b) and a sitespecific recombinase capable of recognizing the two recombinase sites tothe cell; d) allowing a recombination event to occur between the genomeof the cell of a) and the partially human immunoglobulin region,resulting in a replacement of the endogenous immunoglobulin variableregion locus with the partially human immunoglobulin region locus. In aspecific aspect of this method, the partially human immunoglobulinregion comprises V_(H) immunoglobulin gene coding sequences, and furthercomprises i) human D and J gene coding sequences and ii) non-coding Dand J gene and pre-DJ sequences based on the endogenous genome of thenon-human vertebrate host. The recombinase targeting sites areintroduced upstream of the endogenous V_(H) immunoglobulin genes anddownstream of the endogenous D and J gene sequences.

In another specific aspect of this method, the method further providesdeleting the genomic region flanked by the two introduced recombinasesites prior to step c).

The invention provides yet another method for generating a transgenicnon-human vertebrate cell, said method comprising: a) providing anon-human vertebrate cell having a genome that comprises two sets ofsite-specific recombination sites that are not capable of recombiningwith one another, and which flank a portion of an endogenousimmunoglobulin region of the host genome; b) deleting the portion of theendogenous immunoglobulin variable region locus of the genome byintroduction of a recombinase that recognizes a first set ofsite-specific recombination sites, wherein such deletion in the genomeretains the second set of site-specific recombination sites; c)providing a vector comprising a partially human immunoglobulin variableregion locus comprising human coding sequences and non-coding sequencesbased on an endogenous immunoglobulin variable region flanked by thesecond set of site-specific recombination sites; d) introducing thevector of step c) and a site specific recombinase capable of recognizingthe second set of recombinase sites to the cell; and e) allowing arecombination event to occur between the genome of the cell and thepartially human immunoglobulin variable region locus, resulting in areplacement of the endogenous immunoglobulin variable region locus withthe partially human immunoglobulin variable region locus.

Preferably, the non-human mammalian cell for use in each of the abovemethods is a mammalian cell, and more preferably a mammalian embryonicstem (ES) cell. In other aspects, the cell may be an avian cell, andpreferably an avian primordial germ cell.

Once the cells have been subjected to the replacement of the endogenousimmunoglobulin variable region locus, cells comprising the introducedpartially human immunoglobulin variable region are selected andpreferably isolated. In a preferred aspect of the invention, the cellsare non-human mammalian embryonic stem (ES) cells, and the isolated EScell is then utilized to create a transgenic non-human mammal expressingthe partially human immunoglobulin variable region locus. In otheraspects, the cells are primordial germ cells, and the isolated germ cellis then utilized to create a transgenic non-human bird expressing thepartially human immunoglobulin variable region.

In a specific aspect, the invention provides a method for generating anon-human mammalian cell comprising a partially human immunoglobulinregion, said method comprising: a) providing an non-human mammalianembryonic stem (ES) cell having a genome that contains two site-specificrecombination sites that are not capable of recombining with each other,and which flank a portion of the immunoglobulin region; b) providing avector comprising a partially human immunoglobulin region comprisinghuman immunoglobulin variable region coding sequences and non-codingsequences based on an endogenous immunoglobulin variable region, saidpartially human region flanked by the same two site-specificrecombination sites that flank the portion of the immunoglobulin regionin the ES cell; c) bringing the ES cell and said vector into contactwith a site specific recombinase capable of recognizing the tworecombinase sites under appropriate conditions to promote arecombination event resulting in the replacement of the endogenousportion of immunoglobulin region with the partially human immunoglobulinregion in the ES cell.

In another aspect, the invention provides a method for generating atransgenic non-human mammal comprising a partially human immunoglobulinregion, said method comprising: a) introducing one or more site-specificrecombination sites that are not capable of recombining with one anotherinto the genome of a cell of a non-human vertebrate host; b) providing avector comprising a partially human immunoglobulin region having i)human variable coding sequences and ii) non-coding sequences based onthe endogenous variable region, wherein the partially human region isflanked by the same site-specific recombination sites as thoseintroduced to the genome of the host cell of a); c) introducing thevector of step b) and a site specific recombinase capable of recognizingone set of recombinase sites to the cell; d) allowing a recombinationevent to occur between the genome of the cell of a) and the partiallyhuman immunoglobulin region, resulting in a replacement of theendogenous immunoglobulin variable region with the partially humanimmunoglobulin region; e) selecting a cell which comprises the partiallyhuman immunoglobulin region; and f) utilizing the cell to create atransgenic animal comprising the partially human immunoglobulin region.

In a specific aspect, the partially human immunoglobulin regioncomprises human V_(H) coding regions, human D and J gene codingsequences, and non-coding D and J gene and pre-DJ sequences based on theendogenous genome of the non-human vertebrate host. The site-specificrecombination sites are then introduced upstream of an endogenous V_(H)immunoglobulin genes and downstream of the endogenous D and J generegions.

The invention provides another method for generating a transgenicnon-human animal comprising a partially human immunoglobulin region,said method comprising: a) providing a non-human vertebrate cell havinga genome that comprises two sets of site-specific recombination sitesthat are not capable of recombining with one another, and which flank aportion of an endogenous immunoglobulin variable region locus of thehost genome; b) deleting the portion of the endogenous immunoglobulinregion of the host genome by introduction of a recombinase thatrecognizes a first set of site-specific recombination sites, whereinsuch deletion in the genome retains the second set of site-specificrecombination sites; c) providing a vector comprising a partially humanimmunoglobulin variable region locus having human coding sequences andnon-coding sequences based on an endogenous immunoglobulin variableregion locus flanked by the second set of site-specific recombinationsites; d) introducing the vector of step c) and a site specificrecombinase capable of recognizing the second set of site-specificrecombination sites to the cell; e) allowing a recombination event tooccur between the genome of the cell and the partially humanimmunoglobulin variable region, resulting in a replacement of theendogenous immunoglobulin region with the partially human immunoglobulinvariable region; f) selecting a cell which comprises the partially humanimmunoglobulin variable region; and g) utilizing the cell to create atransgenic animal comprising the partially human immunoglobulin variableregion.

The invention provides yet another method for generating a transgenicnon-human mammal comprising a partially human immunoglobulin region,said method comprising: a) providing an non-human mammalian embryonicstem (ES) cell having a genome that contains two site-specificrecombination sites that are not capable of recombining with each other,and which flank a portion of the immunoglobulin region; b) providing avector comprising a partially human immunoglobulin region comprisinghuman variable coding sequences and non-coding sequences based on theendogenous variable gene region, said partially human region flanked bythe same two site-specific recombination sites that flank the portion ofthe immunoglobulin region in the ES cell; c) bringing said ES cell andsaid vector into contact with a site specific recombinase capable ofrecognizing the two recombinase sites under appropriate conditions topromote a recombination event resulting in the replacement of theendogenous portion of immunoglobulin region with the partially humanimmunoglobulin region in the ES cell; d) selecting an ES cell whichcomprises the replaced portion of nucleic acid and using said embryonicstem cell; and e) utilizing the cell to create a transgenic animalcomprising the partially human immunoglobulin variable region locus togenerate a heterozygous partially human animal.

In a specific aspect of the invention, the transgenic non-humanvertebrates are mammals, and preferably the mammals are rodents, e.g., amouse or a rat. In other aspects, the transgenic non-human vertebratesare avian, e.g., a chicken.

It is an object of the invention to provide non-human vertebrate cellsand non-human transgenic mammals expressing an introduced immunoglobulinvariable region locus having human variable region coding sequences andnon-coding sequences based on the endogenous host genome.

Further, it is an object to provide B-cells from transgenic animalswhich are capable of expressing partially human antibodies having humanV_(H) sequences, where such B-cells are immortalized to provide a sourceof a monoclonal antibody specific for a particular antigen.

It is yet another object to provide human variable regions cloned from Bcells for use in the production and/or optimization of antibodies fordiagnostic and therapeutic uses.

It is a further object of the invention to provide hybridoma cells thatare capable of producing partially human monoclonal antibodies havinghuman variable region sequences.

These and other aspects, objects and features are described in moredetail below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a flow chart setting forth one exemplary method fromthe preferred embodiment of the invention.

FIG. 2 is a schematic diagram illustrating the introduction of a firstset of site-specific recombination sites into the genome of a non-humanmammalian cell via a homology targeting vector.

FIG. 3 is another schematic diagram illustrating the introduction of afirst set of site-specific recombination sites into the genome of anon-human mammalian cell via a homology targeting vector.

FIG. 4 is a schematic diagram illustrating the introduction of a secondset of site-specific recombination sites into the genome of a non-humanmammalian cell via a homology targeting vector.

FIG. 5 is a schematic diagram illustrating deletion of the endogenousimmunoglobulin region of the host cell.

FIG. 6 is a schematic diagram illustrating the introduction of thepartially human immunoglobulin region via a site specific targetingvector.

FIG. 7 is a schematic diagram illustrating the introduction of thepartially human immunoglobulin region comprising additional mousesequences using a site specific targeting vector.

FIG. 8 is a schematic diagram illustrating the introduction of thepartially human immunoglobulin region comprising additional mousesequences to a mouse heavy chain region.

FIG. 9 is a schematic diagram illustrating the introduction of thepartially human immunoglobulin region comprising additional mousesequences to a mouse kappa region.

FIG. 10 is a schematic diagram illustrating the introduction of thepartially human immunoglobulin region comprising additional mousesequences to a mouse lambda region.

FIG. 11 is a schematic diagram illustrating the introduction of thepartially human immunoglobulin region comprising a human V_(H) minigenevia a site specific targeting vector.

DEFINITIONS

The terms used herein are intended to have the plain and ordinarymeaning as understood by those of ordinary skill in the art. Thefollowing definitions are intended to aid the reader in understandingthe present invention, but are not intended to vary or otherwise limitthe meaning of such terms unless specifically indicated.

“partially human” as used herein refers to a nucleic acid havingsequences from both a human and a non-human mammal or an animalcomprising a nucleic acid having sequences from both a human and anon-human mammal. In the context of partially human sequences of theinvention, the partially human nucleic acids have sequences of humanimmunoglobulin coding regions and sequences based on the non-codingsequences of the endogenous immunoglobulin region of the non-humanmammal. The term “based on” when used with reference to endogenousnon-coding sequences from a non-human mammal refers to sequences thatcorrespond to the non-coding sequence and share a relatively high degreeof homology with the non-coding sequences of the endogenous loci of thehost mammal, e.g., the mammal from which the ES cell is derived.Preferably, the non-coding sequences share at least an 80%, morepreferably 90% homology with the corresponding non-coding sequencesfound in the endogenous loci of the non-human vertebrate host cell intowhich a partially human molecule comprising the non-coding sequences isbeing introduced.

The term “homology targeting vector” refers to a vector comprising anucleic acid encoding a targeting sequence, a site-specificrecombination site, and optionally a selectable marker gene, which isused to modify immunoglobulin region using homology-mediatedrecombination in a host cell. For example, a homology targeting vectorcan be used in the present invention to introduce a site-specificrecombination site into particular region of a host cell genome.

The term “immunoglobulin variable region” as used herein refers to anucleotide sequence that encodes all or a portion of a variable regionof an antibody molecule or all or a portion of a regulatory nucleotidesequence that controls expression of an antibody molecule.Immunoglobulin regions for heavy chains may include but are not limitedto all or a portion of the V, D, J, and switch regions, includingintrons. Immunoglobulin region for light chains may include but are notlimited to the V and J regions, their upstream flanking sequences,introns, associated with or adjacent to the light chain constant regiongene.

“Site-specific recombination” refers to a process of recombinationbetween two compatible recombination sites including any of thefollowing three events: a) deletion of a preselected nucleic acidflanked by the recombination sites; b) inversion of the nucleotidesequence of a preselected nucleic acid flanked by recombination sites,and c) reciprocal exchange of nucleic acid regions proximate torecombination sites located on different nucleic acid molecules. It isto be understood that this reciprocal exchange of nucleic acid segmentsresults in an integration event if one or both of the nucleic acidmolecules are circular.

The term “targeting sequence” refers to a sequence homologous to DNAsequences in the genome of a cell that flank or occur adjacent to theregion of an immunoglobulin region to be modified. The flanking oradjacent sequence may be within the locus itself or upstream ordownstream of coding sequences in the genome of the host cell. Targetingsequences are inserted into recombinant DNA vectors which are used totransfect such that sequences to be inserted into the cell genome, suchas the sequence of a recombination site, are flanked by the targetingsequences of the vector.

The term “site-specific targeting vector” as used herein refers to avector comprising a nucleic acid encoding a site-specific recombinationsite, a partially human nucleic acid, and optionally a selectable markergene, which is used to modify an endogenous immunoglobulin region in ahost using recombinase-mediated site-specific recombination. Therecombination site of the targeting vector is suitable for site-specificrecombination with another corresponding recombination site which hasbeen inserted into a genomic sequence of the host cell (e.g., via ahomology targeting vector), adjacent to an immunoglobulin region whichis to be modified. Integration of a partially human sequence into arecombination site in an immunoglobulin region results in replacement ofthe endogenous region by the introduced partially human region.

The term “transgene” is used herein to describe genetic material whichhas been or is about to be artificially inserted into the genome of acell, and particularly a cell of a vertebrate host animal. The term“transgene” as used herein refers to a partially human nucleic acid,e.g., a partially human nucleic acid in the form of an expressionconstruct and/or a targeting vector.

By “transgenic animal” is meant a non-human animal, usually a mammal,having an exogenous nucleic acid sequence present as an extrachromosomalelement in a portion of its cells or stably integrated into its germline DNA (i.e., in the genomic sequence of most or all of its cells). Inthe present invention, a partially human nucleic acid is introduced intothe germ line of such transgenic animals by genetic manipulation of, forexample, embryos or embryonic stem cells of the host animal according tomethods well known in the art.

A “vector” includes plasmids and viruses and any DNA or RNA molecule,whether self-replicating or not, which can be used to transform ortransfect a cell.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the techniques described herein may employ, unlessotherwise indicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and sequencing technology,which are within the skill of those who practice in the art. Suchconventional techniques include polymer array synthesis, hybridizationand ligation of polynucleotides, and detection of hybridization using alabel. Specific illustrations of suitable techniques can be had byreference to the examples herein. However, other equivalent conventionalprocedures can, of course, also be used. Such conventional techniquesand descriptions can be found in standard laboratory manuals such asGreen, et al., Eds. (1999), Genome Analysis: A Laboratory Manual Series(Vols. I-IV); Weiner, Gabriel, Stephens, Eds. (2007), Genetic Variation:A Laboratory Manual; Dieffenbach, Dveksler, Eds. (2003), PCR Primer: ALaboratory Manual; Bowtell and Sambrook (2003), DNA Microarrays: AMolecular Cloning Manual; Mount (2004), Bioinformatics: Sequence andGenome Analysis; Sambrook and Russell (2006), Condensed Protocols fromMolecular Cloning: A Laboratory Manual; and Sambrook and Russell (2002),Molecular Cloning: A Laboratory Manual (all from Cold Spring HarborLaboratory Press); Stryer, L. (1995) Biochemistry (4th Ed.) W.H.Freeman, New York N.Y.; Gait, “Oligonucleotide Synthesis: A PracticalApproach” 1984, IRL Press, London; Nelson and Cox (2000), Lehninger,Principles of Biochemistry 3^(rd) Ed., W. H. Freeman Pub., New York,N.Y.; and Berg et al. (2002) Biochemistry, 5^(th) Ed., W.H. FreemanPub., New York, N.Y., all of which are herein incorporated in theirentirety by reference for all purposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a locus” refers toone or more loci, and reference to “the method” includes reference toequivalent steps and methods known to those skilled in the art, and soforth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated by reference for the purpose of describing anddisclosing devices, formulations and methodologies that may be used inconnection with the presently described invention.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in the smaller ranges, andare also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either both of those includedlimits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, well-known features and procedures wellknown to those skilled in the art have not been described in order toavoid obscuring the invention.

THE INVENTION IN GENERAL

In the humoral immune system, a diverse antibody repertoire is producedby combinatorial and junctional diversity of IgH (Igh) and IgL chain(Igl) gene loci in a process termed V(D)J recombination. In thedeveloping B cell, the first recombination event to occur is between oneD and one J gene segment of the heavy chain locus, and the DNA betweenthese two genes is deleted. This D-J recombination is followed by thejoining of one V gene from a region upstream of the newly formed DJcomplex, forming a rearranged V(D)J gene. All other genes between V andD segments of the new V(D)J gene are now deleted from the individual Bcell's genome. This rearranged gene is ultimately expressed on the Bcell surface as an IgH polypeptide, which associates with an IgL to formthe B cell receptor. The murine and human Ig loci are highly complex,spanning regions of approximately 2 Mb, containing several constantregion gene segments, J gene segments, D gene segments and larger numberof variable genes.

The present invention provides non-human vertebrate cells comprising anintroduced partially human nucleic acid comprising coding regions forhuman variable regions and non-coding sequences from the vertebrate hostgenome, e.g., mouse genomic non-coding sequences when the host mammal isa mouse. This partially human nucleic acid allows the transgenic animalto produce a heavy chain repertoire comprising human V_(H) regions,while retaining the regulatory sequences and other elements that can befound within the intervening sequences in a particular host genome thathelp to promote efficient antibody production and antigenic recognition.The present invention comprises the use of a synthetic or recombinantlyproduced partially human region comprising both human coding sequencesand non-human non-coding sequences from a V_(H) locus.

Because the methods of the invention can take advantage of two or moresets of site-specific recombination sites within the engineered genome,the recombination step allows multiple insertions to be made into thepartially human locus.

In preferred aspects of the invention, this partially human region to beintroduced into a host vertebrate cell comprises all or a substantialnumber of the known human V_(H) genes. In some instances, however, itmay be desirable to use a subset of such V_(H) genes, and in specificinstances even as few as one human V_(H) coding sequences may be used inthe cells and the animals of the invention.

The preferred aspects of the invention comprise non-human mammals andmammalian cells comprising a partially human immunoglobulin locus thatcomprises human V_(H) genes and further comprises D and J gene humancoding regions and pre-DJ sequences based on the endogenous genome ofthe non-human mammalian host. In certain aspects, the introducedpartially human region can comprise one or more fully recombined V(D)Jsegments.

In a specific aspect of the invention, the transgenic non-human mammalcomprises an introduced nucleic acid comprising multiple human V_(H)genes with intervening sequences based on the intervening sequences inthe non-human mammalian host loci and human coding regions for human Dand J genes. In a particularly preferred aspect, the partially humannucleic acid comprises the human V_(H) genes, a pre-D region based onthe genome of the non-human mammalian host, e.g., the mouse genome, anda human D and J exons.

In an exemplary embodiment, as set forth in more detail in the Examplessection, the entire endogenous VH immunoglobulin locus of the mouse(including the J558 locus) is deleted, and the VH exons of the J558 VHregion locus of a mouse are replaced with a nucleic acid comprising 44of the human V_(H) genes, which, as a result, are interspersed withnon-coding regions that correspond to the non-coding sequences of mouse.The complete introduced immunoglobulin V_(H) region further compriseshuman D and J exons as well as V_(H) genes. In this aspect, the 10 Kbpre-D region comprises mouse sequences, while the D and J regionscomprise human coding sequences. Preferably, the D and J regions areprovided as a human DJ coding region comprising human D genes and humanJ genes.

The methods of the invention utilize a combination of homologousrecombination and site-specific recombination to create the cells andanimals of the invention. A homology targeting vector is first used tointroduce the site-specific recombination sites into the host mammalgenome at the desired location in the endogenous immunoglobulin loci.Insertion of a site-specific recombination site into a genomic sequencevia homologous recombination of an associated targeting sequence withgenomic DNA in vivo preferably does not modify an amino acid sequence ofthe antibody molecule which is expressed by the transfected cell. Thisapproach maintains the proper transcription and translation of theimmunoglobulin genes which produce the desired antibody after insertionof recombination sites and, optionally, any additional sequence such asa selectable marker gene. However, in some cases it is possible toinsert a recombinase site and other sequences into an immunoglobulinlocus sequence such that an amino acid sequence of the antibody moleculeis altered by the insertion, but the antibody still retains sufficientfunctionality for the desired purpose, and the invention envisionsencompassing such insertions as well.

Exemplary methodologies for homologous recombination are described inU.S. Pat. Nos. 6,689,610; 6,204,061; 5,631,153; 5,627,059; 5,487,992;and 5,464,764, each of which is incorporated by reference in itsentirety.

In specific aspects of the invention, the homology targeting vector canbe utilized to replace certain sequences within the endogenous genome aswell as introducing the site-specific recombination sites and selectablemarkers. For example, the homology targeting used to introduce elements3′ of the V_(H) gene region may be used to replace the mouse pre-D andDJ sequences with the human equivalents.

Site-Specific Recombination

Site-specific recombination differs from general homologousrecombination in that short specific DNA sequences, which are requiredfor the recombinase recognition, are the only sites at whichrecombination occurs. Site-specific recombination requires specializedrecombinases to recognize the sites and catalyze the recombination atthese sites. A number of bacteriophage and yeast derived site-specificrecombination systems, each comprising a recombinase and specificcognate sites, have been shown to work in eukaryotic cells for thepurpose of DNA integration and are therefore applicable for use in thepresent invention, and these include the bacteriophage P1 Cre/lox, yeastFLP-FRT system, and the Dre system of the tyrosine family ofsite-specific recombinases. Such systems and methods of use aredescribed, for example, in U.S. Pat. Nos. 7,422,889; 7,112,715;6,956,146; 6,774,279; 5,677,177; 5,885,836; 5,654,182; and 4,959,317,which are incorporated herein by reference to teach methods of usingsuch recombinases. The recombinase mediated cassette exchange (RMCE)procedure is facilitated by usage of the combination of wild-type andmutant loxP (or FRT etc) sites together with negative selection. It willoccur, however, when only non-mutant sites are used and/or in theabsence of selection. But the efficiency is very low because excisionrather than insertion reactions are favored and (without incorporatingpositive selection) there will be no enrichment for appropriatelymutated cells.

Other systems of the tyrosine family such as bacteriophage lambda Intintegrase, HK2022 integrase, and in addition systems belonging to theseparate serine family of recombinases such as bacteriophage phiC31,R4Tp901 integrases are known to work in mammalian cells using theirrespective recombination sites (Tronche, F. et al. 2002), and are alsoapplicable for use in the present invention.

The methods of the invention preferably utilize site-specificrecombination sites that utilize the same recombinase, but which do notfacilitate recombination between the sites. For example, a Lox P siteand a mutated Lox P site can be integrated into the genome of a host,but introduction of Cre into the host will not cause the two sites tofacilitate recombination; rather, the LoxP site will recombine withanother LoxP site, and the mutated site will only recombine with anotherlikewise mutated LoxP site. Examples of such mutated recombination sitesinclude those that contain a combination of inverted repeats or thosewhich comprise recombination sites having mutant spacer sequences. Forexample, two classes of variant recombinase sites are available toengineer stable Cre-loxP integrative recombination. Both exploitsequence mutations in the Cre recognition sequence, either within the 8bp spacer region or the 13-bp inverted repeats. Spacer mutants such aslox511 (Hoess R H et al., Nucleic Acids Res 1986, 14:2287-2300), lox5171and lox2272 (Lee G and Saito I, Gene 1998, 216:55-65), m2, m3, m7, andm11 (Langer S J et al., Nucleic Acids Res 2002, 30:3067-3077) recombinereadily with themselves but have a markedly reduced rate ofrecombination with the wild-type site. This class of mutants has beenexploited for DNA insertion by recombinase mediated cassette exchange(RMCE) using non-interacting Cre-Lox recombination sites andnon-interacting FLP recombination sites (Baer A and Bode J, Curr OpinBiotechnol 2001, 12:473-480; Albert H et al., Plant J 1995, 7:649-659;Seibler J and Bode J, Biochemistry 1997, 36:1740-1747; Schlake T andBode J, Biochemistry 1994, 33:12746-12751).

Inverted repeat mutants represent the second class of variantrecombinase sites. For example, LoxP sites can contain altered bases inthe left inverted repeat (LE mutant) or the right inverted repeat (REmutant). An LE mutant, lox71, has 5 bp on the 5′ end of the leftinverted repeat that is changed from the wild type sequence to TACCG(Araki K et al, Nucleic Acids Res 1997, 25:868-872). Similarly, the REmutant, lox66, has the five 3′-most bases changed to CGGTA. Invertedrepeat mutants are used for integrating plasmid inserts into chromosomalDNA with the LE mutant designated as the “target” chromosomal loxP siteinto which the “donor” RE mutant recombines. Post-recombination, loxPsites are located in cis, flanking the inserted segment. The mechanismof recombination is such that post-recombination one loxP site is adouble mutant (containing both the LE and RE inverted repeat mutations)and the other is wild type (Lee L and Sadowski PD, Prog Nucleic Acid ResMol Biol 2005, 80:1-42; Lee L and Sadowski PD, J Mol Biol 2003,326:397-412). The double mutant is sufficiently different from thewild-type site that it is unrecognized by Cre recombinase and theinserted segment is not excised.

In certain aspects, site-specific recombination sites can be introducedinto introns, as opposed to coding nucleic acid regions or regulatorysequences. This may avoid inadvertently disrupting any regulatorysequences or coding regions necessary for proper antibody expressionupon insertion of site-specific recombination sites into the genome ofthe animal cell.

Introduction of the site-specific recombination sites may be achieved byconventional homologous recombination techniques. Such techniques aredescribed in references such as e.g., Sambrook and Russell (2001)(Molecular cloning: a laboratory manual 3rd edn (Cold Spring Harbor,N.Y.: Cold Spring Harbor Laboratory Press) and Nagy, A. (2003).(Manipulating the mouse embryo: a laboratory manual, 3rd edn (ColdSpring Harbor, N.Y.: Cold Spring Harbor Laboratory Press). GeneticRecombination: Nucleic acid, Homology (biology), Homologousrecombination, Non-homologous end joining, DNA repair, Bacteria,Eukaryote, Meiosis, Adaptive immune system, V(D)J recombination byFrederic P. Miller, Agnes F. Vandome, and John McBrewster(Paperback—Dec. 23, 2009).

Specific recombination into the genome can be facilitated using vectorsdesigned for positive or negative selection as known in the art. Inorder to facilitate identification of cells that have undergone thereplacement reaction, an appropriate genetic marker system may beemployed and cells selected by, for example use of a selection medium.However, in order to ensure that the genome sequence is substantiallyfree of extraneous nucleic acid sequences at or adjacent to the two endpoints of the replacement interval, desirably the marker system/gene canbe removed following selection of the cells containing the replacednucleic acid.

In one preferred aspect of the methods of the present invention, cellsin which the replacement of all or part of the endogenous immunoglobulinhas taken place are negatively selected upon exposure to a toxin ordrug. For example, cells that retain expression of HSV-TK can beselected through use of appropriate use of nucleoside analogues such asgancyclovir. In another aspect of the invention, cells comprising thedeletion of the endogenous immunoglobulin region may be positivelyselected by use of a marker gene, which can optionally be removed fromthe cells following or as a result of the recombination event. Apositive selection system that may be used is based on the use of twonon-functional portions of a marker gene, such as HPRT, that are broughttogether through the recombination event. These two portions are broughtinto functional association upon a successful replacement reaction beingcarried out and wherein the functionally reconstituted marker gene isflanked on either side by further site-specific recombination sites(which are different to the site-specific recombination sites used forthe replacement reaction), such that the marker gene can be excised fromthe genome, using an appropriate site-specific recombinase.

The recombinase may be provided as a purified protein, or a constructtransiently expressed within the cell in order to provide therecombinase activity. Alternatively, the cell may be used to generate atransgenic animal, which may be crossed with an animal which expressessaid recombinase, in order to produce progeny which lack the marker geneand associated recombination sites.

Generation of Transgenic Animals

In specific aspects, the invention provides methods for the creation oftransgenic animals comprising the introduced partially humanimmunoglobulin region.

In one aspect, the host cell utilized for replacement of the endogenousimmunoglobulin genes is an embryonic stem (ES) cell, which can then beutilized to create a transgenic mammal. Thus, in accordance with oneaspect, the methods of the invention further comprise: isolating anembryonic stem cell which comprises the introduced partially humanimmunoglobulin region and using said ES cell to generate a transgenicanimal that contains the replaced partially immunoglobulin locus.

In another example, the transgenic animal is avian, and the animal isproduced using primordial germ cells. Thus, in accordance with anotheraspect, the methods of the invention further comprise: isolating aprimordial germ cell which comprises the introduced partially humanimmunoglobulin region and using said germ cell to generate a transgenicanimal that contains the replaced partially immunoglobulin locus.Methods for production of such transgenic avians are disclosed, e.g., inU.S. Pat. Nos. 7,323,618 and 7,145,057, which are incorporated herein byreference.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. It will be appreciated by personsskilled in the art that numerous variations and/or modifications may bemade to the invention as shown in the specific embodiments withoutdeparting from the spirit or scope of the invention as broadlydescribed. The present embodiments are, therefore, to be considered inall respects as illustrative and not restrictive.

Efforts have been made to ensure accuracy with respect to terms andnumbers used (e.g., vectors, amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees centigrade,and pressure is at or near atmospheric.

The examples illustrate targeting by both a 5′ vector and a 3′ vectorthat flank a site of recombination and introduction of synthetic DNA. Itwill be apparent to one skilled in art upon reading the specificationthat the 5′ vector targeting can take place first followed by the 3′, orthe 3′ vector targeting can take place followed by the 5′ vector. Insome circumstances, targeting can be carried out simultaneously withdual detection mechanisms.

Example 1 Introduction of a Partially Human Immunoglobulin Region intothe V_(H) Gene Locus of a Mouse Genome

An exemplary method for replacing a portion of a mammalian genome withpartially human immunoglobulin region is illustrated in FIGS. 1-6. FIG.1 shows a flow chart illustrating the different steps of this exemplaryaspect of the methods of the invention. This method provides introducinga first site-specific recombination site into the mammalian genome,which may be introduced either 5′ or 3′ of an endogenous V_(H) region ofthe mammalian genome. This is then followed by the introduction 102 of asecond site-specific recombination site into the mammalian genome, whichin combination with the first site-specific recombination site flanksthe endogenous immunoglobulin region. The flanked endogenous region isdeleted 104 and a synthetic nucleic acid comprising both human andnon-human sequences is introduced 106 via recombinase mediated exchange.

An exemplary method illustrating the introduction of a partially humanmouse-human immunoglobulin region into the genomic locus of a mouse EScell is illustrated in more detail in FIGS. 2-6. In FIG. 2, a homologytargeting vector 201 is provided comprising a puromycinphosphotransferase-thymidine kinase fusion protein (puroΔTK) 203 flankedby two different recombinase recognition sites, e.g., FRT 207 and loxP205, for Flp and Cre, and modified sites e.g., for FRT 209 and loxP 211,which have the inability to recombine with the unmodified sites 207 and205, respectively. The targeting vector comprises a human diphtheriatoxin receptor (hDTR) cDNA 217 for use in negative selection of cellsexpressing the introduced construct in future steps. The targetingvector also optionally comprises a visual marker such as a fluorescentgreen protein (GFP) (not shown). The regions 213 and 215 are homologousto the 5′ and 3′ portions, respectively, of a contiguous region 223 inthe endogenous mouse locus that is 5′ of the genomic region 219comprising the mouse endogenous V_(H) genes. The homology targetingvector 201 is introduced 202 to the mouse ES cell, which has animmunoglobulin region 229 comprising endogenous V_(H) genes 219, thepre-D region 221, the J gene region 225 and the constant gene region 227of the immunoglobulin region. The site specific recombination sites andthe hDTR cDNA 217 of the homology targeting vector 201 is integrated 204into the mouse genome 5′ of the mouse endogenous V_(H) gene region.

FIG. 3 illustrates effectively the same approach as FIG. 2, except thatan additional set of site-specific recombination sites is added, e.g., aRox site 331 and a modified Rox site 333 for use with the Drerecombinase. In FIG. 3 a homology targeting vector 301 is providedcomprising a puromycin phosphotransferase-thymidine kinase fusionprotein 303 flanked by recombinase recognition sites FRT 307, loxP 305,and Rox 331 and modified sites for FRT 309 loxP 311 and Rox 333, whichhave the inability to recombine with the unmodified sites 307, 305 and331, respectively. The targeting vector also comprises a humandiphtheria toxin receptor (hDTR) cDNA 317. The regions 313 and 315 arehomologous to the 5′ and 3′ portions, respectively, of a contiguousregion 323 in the endogenous mouse locus that is 5′ of the genomicregion 319 comprising the mouse endogenous V_(H) genes. The homologytargeting vector 301 is introduced 302 to the mouse immunoglobulinregion 329, which comprises the endogenous V_(H) genes 319, the pre-Dregion 321, the J gene region 325 and the constant gene region 327 ofthe immunoglobulin region. The site specific recombination sites and thehDTR cDNA 317 of the homology targeting vector 301 is integrated 304into the mouse genome 5′ of the mouse endogenous V_(H) gene region.

As illustrated in FIG. 4, a second homology targeting vector 401 isprovided comprising a hypoxanthinephosphoribosyltransferase (HPRT)mini-gene 435 and a neomycin resistance gene 437 and recombinaserecognition sites FRT 407 and loxP 405, for Flp and Cre, which have theability to recombine with FRT 407 and loxP 405 sites integrated from thefirst homology targeting vector. The regions 431 and 433 are homologousto the 5′ and 3′ portions, respectively, of a contiguous region 441 inthe endogenous mouse locus that is 3′ of the genomic region comprisingthe mouse endogenous V_(H), D and J genes and 5′ of the constant generegion. The homology targeting vector 401 is introduced 402 to themodified mouse immunoglobulin region, which comprises the endogenousV_(H) genes 419, the pre-D region 421, the J gene region 425 and theconstant gene region 427. The site specific recombination sites and theHPRT mini-gene 435 and a neomycin resistance gene 437 of the homologytargeting vector 401 is integrated 404 into the mouse genome 5′ of themouse endogenous V_(H) gene region.

Once the recombination sites are introduced to the host mammal's genome,the endogenous region of the immunoglobulin domain is then subject torecombination by introducing one of the recombinases corresponding tothe site-specific recombination sites in the genome, in this exampleeither FLP or Cre. As illustrated in FIG. 5, when FLP is introduced 502,the region containing the site-specific recombination sites (509, 511,507 and 505) and the puroΔTK gene 503 are retained, with an additionalFLP recombination site 507 now present 3′ of the other two recombinationsites 507 and 505. The region 3′ of the recombination sites—includingthe hDTR 517, the endogenous immunoglobulin domain (519, 521, 525), andthe HPRT 527 and Neo 529 genes introduced using the second homologytargeting vector are deleted. When Cre is used for recombinase-mediateddeletion 504, the area of deletion is the same, but only one sitespecific recombination site 507 remains directly 3′ of the puroΔTK gene.The procedure depends on the second targeting having occurred on thesame chromosome rather than on its homolog (i.e., in cis rather than intrans). If the targeting occurs in trans, the cells will not besensitive to negative selection after Cre recombination.

The primary screening for deletion of the endogenous immunoglobulinregion can be carried out by Southern blot, or with primary polymerasechain reaction (PCR) screens supported by secondary screens withSouthern and/or loss-of-native-allele qPCR screens. HPRT will allow for(6-thioguanine-dependent) negative selection in HPRT-deficient ES cells.ES cells with a deleted immunoglobulin region can be selected bynegative selection using the hDTR gene.

FIG. 6 illustrates the introduction of the partially human sequence tothe modified mouse genome. A site-specific targeting vector 629comprising the partially human immunoglobulin region 610 to beintroduced to the mammalian host genome is introduced 602 to the genomicregion 601 with the deleted endogenous immunoglobulin region comprisingthe site-specific recombination sites (609, 611, 607 and 605) and thepuroΔTK gene 603. The site-specific targeting vector comprised apartially human immunoglobulin region comprising i) a V_(Hregion) 619comprising 44 human V_(H) coding regions and intervening sequences basedon the mouse genome endogenous sequences; ii) a 10 kb pre-DJ region 621comprising mouse sequence; and iii) a DJ region 625 comprising human Dand J gene coding regions and intervening sequences based on the mousegenome endogenous sequences. The partially human immunoglobulin regionis flanked by recombination sites (609, 611, 605 and 607) that willallow recombination with the modified endogenous locus. Uponintroduction of the appropriate recombinase 604, the partially humanimmunoglobulin region is integrated into the genome upstream of theconstant gene region 627.

The primary screening for introduction of the partially humanimmunoglobulin region can be carried out by Southern blot, or withprimary PCR screens supported by secondary screens with Southern and/orloss-of-native-allele qPCR screens. The deletion of the HPRT gene 605 aspart of the recombination event will allow identification of the cellsthat did not undergo the recombination event using(6-thioguanine-dependent) negative selection.

Example 2 Introduction of a Partially Human Immunoglobulin Region into aMouse Genome

In certain aspects, the partially human immunoglobulin region willcomprise the elements as described in Example 1, but with additionalsequences e.g., sequences strategically added to introduce additionalregulatory sequences, to ensure desired spacing within the introducedimmunoglobulin region, to ensure that certain coding sequences are inadequate juxtaposition with other sequences adjacent to the replacedimmunoglobulin region, and the like. FIG. 7 illustrates the introductionof a second exemplary partially human sequence to the modified mousegenome as produced in FIGS. 2-5 and described in Example 1 above.

A site-specific targeting vector 729 comprising the partially humanimmunoglobulin region 710 to be introduced to the mammalian host genomeis introduced 702 to the genomic region 701 with the deleted endogenousimmunoglobulin region comprising the site-specific recombination sites(709, 711, 707 and 705) and the puroΔTK gene 703. The site-specifictargeting vector comprised a partially human immunoglobulin regioncomprising i) a V_(H) region 719 comprising 1-43 human V_(H) codingregions and intervening sequences based on the mouse genome endogenoussequences; ii) a 10 kb pre-DJ region 721 comprising mouse sequence; iii)a DJ region 725 comprising human D and J coding regions and interveningsequences based on the mouse genome endogenous sequences; and iv) amouse non-functional J_(H) gene region. The partially humanimmunoglobulin region is flanked by recombination sites 709, 711, 705and 707) that will allow recombination with the modified endogenouslocus. Upon introduction of the appropriate recombinase 704, thepartially human immunoglobulin region is integrated into the genomeupstream of the constant gene region 727.

As described in Example 1, the primary screening for introduction of thepartially human immunoglobulin region can be carried out by Southernblot, or with primary PCR screens supported by secondary screens withSouthern and/or loss-of-native-allele qPCR screens. The deletion of theHPRT gene 705 as part of the recombination event will allowidentification of the cells that did not undergo the recombination eventusing (6-thioguanine-dependent) negative selection.

Example 3 Introduction of a Partially Human Immunoglobulin Region intothe Immunoglobulin Heavy Chain Gene Locus of a Mouse Genome

A method for replacing a portion of a mammalian genome with partiallyhuman immunoglobulin region is illustrated in FIG. 8. This method usedintroduction of a first site-specific recombination site into themammalian genome followed by the introduction of a second site-specificrecombination site into the mammalian genome. The two sites flanked theentire cluster of V_(H), D_(H) and J_(H) region gene segments. Theflanked endogenous region was deleted using the relevant site-specificrecombinase, as described herein.

The targeting vectors 803, 805 employed for introducing thesite-specific recombinase sites on either side of the V_(H), D_(H) andJ_(H) region gene segment cluster in the wild-type mouse immunoglobulinregion 801 included an additional site-specific recombinase site thathas been modified so that it is still recognized efficiently by therecombinase, but will not recombine with unmodified sites. This site waspositioned in the targeting vector such that after deletion of theV_(H), D_(H) and J_(H) region gene segment cluster it could be used fora second site specific recombination event in which a non-native pieceof DNA is moved into the modified V_(H) locus. The process of moving theDNA into the locus using the site-specific recombinase is referred to as“recombinase-mediated cassette exchange”. In this example, thenon-native DNA was a synthetic nucleic acid comprising both human andnon-human sequences.

Two gene targeting vectors were constructed to accomplish the processjust outlined. One of the vectors 803 comprised mouse genomic DNA takenfrom the 5′ end of the locus, upstream of the most distal variableregion gene segment. The other vector 805 comprised mouse genomic DNAtaken from within the locus in the vicinity of the J region genesegments.

The key features of the 5′ vector 803 in order from 5′ to 3′ were asfollows: a gene encoding the diphtheria toxin A (DTA) subunit undertranscriptional control of a modified herpes simplex virus type Ithymidine kinase gene promoter coupled to two mutant transcriptionalenhancers from the polyoma virus; 4.5 Kb of mouse genomic DNA mappingupstream of the most distal variable region gene segment in the heavychain locus; a J region gene segment (disabled); an FRT recognitionsequence for the Flp recombinase; a piece of genomic DNA containing themouse Polr2a gene promoter; a translation initiation sequence(methionine codon embedded in a “Kozak” consensus sequence); a mutatedloxP recognition sequence (known as a lox5171 site) for the Crerecombinase; a transcription termination/polyadenylation sequence; aloxP recognition sequence for the Cre recombinase; a gene encoding afusion protein comprised of a protein conferring resistance to puromycinfused to a truncated form of the thymidine kinase (pu-TK) undertranscriptional control of the promoter from the mouse phosphoglyceratekinase 1 gene; and 3 Kb of mouse genomic DNA mapping close to the 4.5 Kbsequence in the vector and arranged in the native relative orientation.

The key features of the 3′ vector 805 in order from 5′ to 3′ were asfollows: a gene encoding the diphtheria toxin A (DTA) subunit undertranscriptional control of a modified herpes simplex virus type Ithymidine kinase gene promoter coupled to two mutant transcriptionalenhancers from the polyoma virus; 3.7 Kb of mouse genomic DNA containingthe mouse J region gene segments oriented such that the end of theregion that maps closest to the heavy chain variable region genesegments was closest to the DTA gene in the vector; a minigene encodingthe human hypoxanthine-guanine phosphoribosyl transferase (HPRT) undertranscriptional control of the mouse Polr2a gene promoter; a neomycinresistance gene under the control of the mouse phosphoglycerate kinase 1gene promoter; a loxP recognition sequence for the Cre recombinase; and2.1 Kb of mouse genomic DNA that maps immediately downstream in thegenome of the 3.7 Kb fragment with the two fragments oriented in thesame configuration as in the mouse genome.

Mouse embryonic stem (ES) cells (derived from C57B1/6NTac mice) weretransfected by electroporation with the 3′ vector 805 according towidely used procedures. Prior to electroporation, the vector DNA waslinearized with the NotI restriction enzyme. The transfected cells wereplated and after ≧24 hours they were placed under drug selection usingthe neomycin analogue G418. Colonies of drug-resistant ES cells werephysically extracted from their plates after they became visible to thenaked eye over a week later. These picked colonies were disaggregated,re-plated in micro-well plates, and cultured for several days.Thereafter, each of the clones of cells was divided such that some ofthe cells could be frozen as an archive, and the rest used for isolationof DNA for analytical purposes.

DNA from the ES cell clones was screened by PCR using a widely usedgene-targeting assay design. Four assays were used, and in each case oneof the PCR oligonucleotide primer sequences mapped outside the region ofidentity shared between the 3′ vector 805 and the genomic DNA, while theother mapped within the novel DNA between the two arms of genomicidentity in the vector (i.e., in the HPRT or neo gene elements).According to the standard design, these assays were designed to detectpieces of DNA that would only be present in clones of cells derived fromtransfected cells that had undergone fully legitimate homologousrecombination between the 3′heavy targeting vector and the genome.

Two separate transfections were performed with the 3′ vector 805. Thefirst of these yielded a total of two positive clones from approximately300 clones screened using the four PCR assays. The second yielded atotal of six positive clones, also from approximately 300 clonesscreened. A total of six PCR-positive clones from the two transfectionswere selected for expansion followed by further analysis using Southernblot assays.

The Southern blot assays are performed according to widely usedprocedures using three probes and genomic DNA digested with multiplerestriction enzymes chosen so that the combination of probes and digestsallow the structure of the targeted locus in the clones to be identifiedas properly modified by homologous recombination. One of the probes mapsto DNA sequence flanking one side of the region of identity sharedbetween the 3′heavy targeting vector and the genomic DNA; a second probemaps outside the region of identity but on its other side; and the thirdprobe maps within the within the novel DNA between the two arms ofgenomic identity in the vector (i.e., in the HPRT or neo gene elements).

The six PCR-positive clones of ES cells are analyzed karyotypicallyusing an in situ fluorescence hybridization procedure designed todistinguish the most commonly arising chromosomal aberrations that arisein mouse ES cells. Clones with such aberrations are excluded fromfurther use. ES cell clones that are judged to have the expected correctgenomic structure based on the Southern blot data, and that also do nothave detectable chromosomal aberrations based on the karyotype analysis,are selected for further use.

Acceptable clones are modified with the 5′ vector 803 using proceduresand screening assays that are essentially identical in design to thoseused with the 3′ vector 805 except puromycin selection is used insteadof G418/neomycin selection. The PCR assays, probes and digests are alsotailored to match the genomic region being modified by the 5′ vector805.

Clones of ES cells that have been mutated in the expected fashion byboth the 3′heavy and the 5′heavy vectors, i.e., doubly-targeted cellscarrying both engineered mutations are isolated following vectortargeting. The clones must have undergone gene targeting on the samechromosome, as opposed to homologous chromosomes (i.e., the engineeredmutations created by the targeting vectors must be in cis on the sameDNA strand rather than in trans on separate homologous DNA strands).Clones with the cis arrangement of mutations are distinguished fromthose with the trans arrangement by analytical procedures such asfluorescence in situ hybridization of metaphase spreads using probesthat hybridize to the novel DNA present in the two gene targetingvectors between their arms of genomic identity. The two types of clonescan also be distinguished from one another by transfecting them with avector expressing the Cre recombinase and then comparing the number ofcolonies that survive gancyclovir selection against the thymidine kinasegene introduced by the 5′ vector 803 and by analyzing the drugresistance phenotype of the surviving clones by a “sibling selection”screening procedure in which some of the cells from the clone are testedfor resistance to puromycin or G418/neomycin. Cells with the cisarrangement of mutations are expected to yield approximately 10³ moregancyclovir-resistant clones than cells with the trans arrangement inthis type of experiment. The majority of the resulting cis-derivedgancyclovir-resistant clones are also be sensitive to both puromycin andG418/neomycin, in contrast to the trans-derived gancyclovir-resistantclones, which should retain resistance to both drugs. Doubly-targetedclones of cells with the cis-arrangement of engineered mutations in theheavy chain locus are selected for further use.

The doubly targeted clones of cells are transfected with a vectorexpressing the Cre recombinase and the transfected cells subsequentlyare placed under gancyclovir selection, as in the analytical experimentsummarized above. Gancyclovir-resistant clones of cells are isolated andanalyzed by PCR and Southern blot for the presence of the expecteddeletion between the two engineered mutations created by the 5′heavy andthe 3′heavy targeting vectors. In these clones, the Cre recombinasecauses a recombination 802 to occur between the loxP sites introducedinto the heavy chain locus by the two vectors to create the constructshown at 807. Because the loxP sites are arranged in the same relativeorientations in the two vectors, recombination results in excision of acircle of DNA comprising the entire genomic interval between the twoloxP sites. The circle does not contain an origin of replication andthus will not be replicated during mitosis and will therefore be lostfrom the clones of cells as they undergo clonal expansion. The resultingclones carry a deletion of the DNA that was originally between the twoloxP sites.

ES cell clones carrying the deletion of sequence in one of the twohomologous copies of their immunoglobulin heavy chain locus areretransfected 804 with a Cre recombinase expression vector together witha piece of DNA 809 comprising a partially human immunoglobulin heavychain locus containing V, D and J region gene segments. The key featuresof this piece of DNA 809 are the following: a lox5171 site; a neomycinresistance gene open reading frame (lacking the initiator methioninecodon, but in-frame and contiguous with an uninterrupted open readingframe in the lox5171 site); a transcription termination/polyadenylationsequence; an FRT site; an array of 44 human heavy chain variable regiongene segments, each comprised of human coding sequences embedded inmouse noncoding sequences; a 7.5 Kb piece of genomic DNA fromimmediately upstream of the cluster of D region gene segments in themouse heavy chain locus; a 58 Kb piece of DNA containing the human D andJ region gene segments; a loxP site in opposite relative orientation tothe lox5171 site.

The transfected clones are placed under G418 selection, which enrichesfor clones of cells that have undergone a recombinase-mediated cassetteexchange process in which the partially human donor DNA 809 (SEQ IDNO:1) is integrated in its entirety into the deleted immunoglobulinheavy chain locus between the loxP and lox5171 sites to create the DNAregion illustrated at 811. The remaining elements from the 5′ vector 803are removed via FLP-mediated recombination 806 resulting in the finalhumanized locus as shown at 813.

G418-resistant ES cell clones are analyzed by PCR and Southern blot todetermine if they have undergone the expected recombinase-mediatedcassette exchange process without unwanted rearrangements or deletions.Clones that have the expected genomic structure are selected for furtheruse.

ES cell clones carrying the partially human immunoglobulin heavy chainDNA 813 in the mouse heavy chain locus are microinjected into mouseblastocysts from strain DBA/2 to create partially ES cell-derivedchimeric mice according to standard procedures. Male chimeric mice withthe highest levels of ES cell-derived contribution to their coats willbe selected for mating to female mice. The female mice of choice herewill be of C57B1/6NTac strain, and will also carry a transgene encodingthe Flp recombinase that is expressed in their germline. Offspring fromthese matings are analyzed for the presence of the partially humanimmunoglobulin heavy chain locus, and for loss of the FRT-flankedneomycin resistance gene that was created in the recombinase-mediatedcassette exchange step. Mice that carry the partially human locus willbe used to establish a colony of mice.

Example 4 Introduction of a Partially Human Immunoglobulin Region intothe Immunoglobulin Kappa Chain Gene Locus of a Mouse Genome

Another method for replacing a portion of a mammalian genome withpartially human immunoglobulin region is illustrated in FIG. 9. Thismethod provides introducing a first site-specific recombination siteinto the mammalian genome, which may be introduced either 5′ or 3′ ofthe main cluster of V_(K) and J_(K) region gene segments of themammalian genome, followed by the introduction of a second site-specificrecombination site into the mammalian genome, which in combination withthe first site-specific recombination site flanks the entire cluster ofV_(K) and J_(K) region gene segments. The flanked endogenous region canthen be deleted and replaced using the relevant site-specificrecombinase.

The targeting vectors employed for introducing the site-specificrecombinase sites on either side of the V_(K) and J_(K) region genesegment cluster 901 also include an additional site-specific recombinasesite that has been modified so that it is still recognized efficientlyby the recombinase, but will not recombine with unmodified sites. Thissite is positioned in the targeting vector such that after deletion ofthe V_(K) and J_(K) region gene segment cluster it can be used for asecond site specific recombination event in which a non-native piece ofDNA is moved into the modified V_(K) locus via recombinase-mediatedcassette exchange. In this example, the non-native DNA is a syntheticnucleic acid comprising both human and non-human sequences.

Two gene targeting vectors were constructed to accomplish the processjust outlined. One of the vectors 903 was comprised of mouse genomic DNAtaken from the 5′ end of the locus, upstream of the most distal variableregion gene segment. The other vector 905 was comprised of mouse genomicDNA taken from within the locus in the vicinity of the J region genesegments.

The key features of the 5′ vector 903 were as follows: a gene encodingthe diphtheria toxin A (DTA) subunit under transcriptional control of amodified herpes simplex virus type I thymidine kinase gene promotercoupled to two mutant transcriptional enhancers from the polyoma virus;6 Kb of mouse genomic DNA mapping upstream of the most distal variableregion gene segment in the kappa chain locus; an FRT recognitionsequence for the Flp recombinase; a piece of genomic DNA containing themouse Polr2a gene promoter; a translation initiation sequence(methionine codon embedded in a “Kozak” consensus sequence); a mutatedloxP recognition sequence (known as a lox5171 site) for the Crerecombinase; a transcription termination/polyadenylation sequence; aloxP recognition sequence for the Cre recombinase; a gene encoding afusion protein comprised of a protein conferring resistance to puromycinfused to a truncated form of the thymidine kinase (pu-TK) undertranscriptional control of the promoter from the mouse phosphoglyceratekinase 1 gene; 2.5 Kb of mouse genomic DNA mapping close to the 6 Kbsequence in the vector and arranged in the native relative orientation.

The key features of the 3′ vector 905 were as follows: a gene encodingthe diphtheria toxin A (DTA) subunit under transcriptional control of amodified herpes simplex virus type I thymidine kinase gene promotercoupled to two mutant transcriptional enhancers from the polyoma virus;6 Kb of mouse genomic DNA taken from the vicinity of the kappa locus Jregion gene segments oriented such that end of the fragment that mapsclosest to the kappa variable region gene segments was closest to theDTA gene in the vector; a minigene encoding the humanhypoxanthine-guanine phosphoribosyl transferase (HPRT) undertranscriptional control of the mouse Polr2a gene promoter; a neomycinresistance gene under the control of the mouse phosphoglycerate kinase 1gene promoter; a loxP recognition sequence for the Cre recombinase; 3.6Kb of mouse genomic DNA that maps immediately downstream in the genomeof the 6 Kb fragment also included in the vector, with the two fragmentsoriented in the same relative way as in the mouse genome.

Mouse embryonic stem (ES) cells derived from C57B1/6NTac mice weretransfected by electroporation with the 3′ vector 905 according towidely used procedures. Prior to electroporation, the vector DNA waslinearized with the NotI restriction enzyme. The transfected cells wereplated and after ≧24 hours they were placed under drug selection usingthe neomycin analogue G418. Colonies of drug-resistant ES cells werephysically extracted from their plates after they became visible to thenaked eye over a week later. These picked colonies were disaggregated,re-plated in micro-well plates, and cultured for several days.Thereafter, each of the clones of cells was divided such that some ofthe cells could be frozen as an archive, and the rest used for isolationof DNA for analytical purposes.

DNA from the ES cell clones was screened by PCR using a widely usedgene-targeting assay design. Four assays were used, and in each case oneof the PCR oligonucleotide primer sequences mapped outside the region ofidentity shared between the 3′ vector 905 and the genomic DNA 901, whilethe other mapped within the novel DNA between the two arms of genomicidentity in the vector (i.e., in the HPRT or neo gene elements).According to the standard design, these assays were designed to detectpieces of DNA that would only be present in clones of cells derived fromtransfected cells that had undergone fully legitimate homologousrecombination between the 3′ vector 905 and the genome

One transfection was performed with the 3′ vector 905 and this yielded atotal of seventeen positive clones from approximately 300 clonesscreened using the four PCR assays.

A total of six PCR-positive clones from the transfection were selectedfor expansion followed by further analysis using Southern blot assays.The Southern blot assays were performed according to widely usedprocedures; they involved three probes and genomic DNA digested withmultiple restriction enzymes chosen so that the combination of probesand digests allowed for conclusions to be drawn about the structure ofthe targeted locus in the clones and whether it had been properlymodified by homologous recombination. One of the probes mapped to DNAsequence flanking one side of the region of identity shared between the3′kappa targeting vector and the genomic DNA; a second probe also mappedoutside the region of identity but on its other side; and the thirdprobe mapped within the within the novel DNA between the two arms ofgenomic identity in the vector (i.e., in the neo gene). The Southernblot identified the presence of the expected Eco911-generated fragmentof DNA corresponding to the correctly mutated (by homologousrecombination with the 3′kappa targeting vector) part of the kappa locusas detected by one of the external probes and by the neomycin probe(data not shown). The external probe detects the mutant fragment andalso a wild-type fragment from the nonmutant copy of the immunoglobulinkappa locus on the homologous chromosome.

The six PCR-positive clones of ES cells were also analyzedkaryotypically using an in situ fluorescence hybridization proceduredesigned to distinguish the most commonly arising chromosomalaberrations that arise in mouse ES cells. One clone with such anaberration was excluded from further use. Two karyoptypically normalclones that were judged to have the expected correct genomic structurebased on the Southern blot data were selected for further use.

The two clones are modified with the 5′ vector 903 using procedures andscreening assays that are essentially identical in design to those usedwith the 3′ vector 905, except puromycin selection is used instead ofG418/neomycin selection, and the protocols are tailored to match thegenomic region modified by the 5′ vector 903. The goal of the 5′ vector903 transfection experiments is to isolate clones of ES cells that havebeen mutated in the expected fashion by both the 3′ vector 905 and the5′ vector 903, i.e., doubly-targeted cells carrying both engineeredmutations. In these clones, the Cre recombinase causes a recombination902 to occur between the loxP sites introduced into the kappa locus bythe two vectors to create the construct shown at 907.

Further, the clones must have undergone gene targeting on the samechromosome, as opposed to homologous chromosomes, i.e., the engineeredmutations created by the targeting vectors must be in cis on the sameDNA strand rather than in trans on separate homologous DNA strands.Clones with the cis arrangement of mutations are distinguished fromthose with the trans arrangement by analytical procedures such asfluorescence in situ hybridization of metaphase spreads using probesthat hybridize to the novel DNA present in the two gene targetingvectors between their arms of genomic identity. The two types of clonescan also be distinguished from one another by transfecting them with avector expressing the Cre recombinase and comparing the number ofcolonies that survive gancyclovir selection against the thymidine kinasegene introduced by the 5′ vector 903 and by analyzing the drugresistance phenotype of the surviving clones by a “sibling selection”screening procedure in which some of the cells from the clone are testedfor resistance to puromycin or G418/neomycin.

Cells with the cis arrangement of mutations are expected to yieldapproximately 10³ more gancyclovir-resistant clones than cells with thetrans arrangement in this type of experiment. The majority of theresulting cis-derived gancyclovir-resistant clones should also besensitive to both puromycin and G418/neomycin, in contrast to thetrans-derived gancyclovir-resistant clones, which should retainresistance to both drugs. Clones of cells with the cis-arrangement ofengineered mutations in the kappa chain locus are selected for furtheruse.

The doubly targeted clones of cells are transfected with a vectorexpressing the Cre recombinase and the transfected cells willsubsequently be placed under gancyclovir selection, as in the analyticalexperiment summarized above. Gancyclovir-resistant clones of cells areisolated and analyzed by PCR and Southern blot for the presence of theexpected deletion between the two engineered mutations created by the 5′vector 903 and the 3′ vector 905. In these clones, the Cre recombinasehas caused a recombination to occur between the loxP sites introducedinto the kappa chain locus by the two vectors. Because the loxP sitesare arranged in the same relative orientations in the two vectors,recombination results in excision of a circle of DNA comprising theentire genomic interval between the two loxP sites. The circle will notcontain an origin of replication and thus will not be replicated duringmitosis and will therefore be lost from the clones of cells as theyundergo clonal expansion. The resulting clones carry a deletion of theDNA that was originally between the two loxP sites. Clones that have theexpected deletion will be selected for further use.

The ES cell clones carrying the deletion of sequence in one of the twohomologous copies of their immunoglobulin kappa chain locus, will beretransfected 904 with a Cre recombinase expression vector together witha piece of DNA 909 comprising a partially human immunoglobulin kappachain locus containing V and J region gene segments. The key features ofthis piece of DNA (referred to as “K-K”) (SEQ ID NO:2) are thefollowing: a lox5171 site; a neomycin resistance gene open reading frame(lacking the initiator methionine codon, but in-frame and contiguouswith an uninterrupted open reading frame in the lox5171 site); atranscription termination/polyadenylation sequence; an FRT site; anarray of 39 human kappa variable region gene segments, each comprised ofhuman coding sequences embedded in mouse noncoding sequences; a 13.5 Kbpiece of genomic DNA from immediately upstream of the cluster of J kapparegion gene segments in the mouse kappa chain locus; a 2 Kb piece of DNAcontaining 5 human J region gene segments embedded in mouse noncodingDNA; a loxP site in opposite relative orientation to the lox5171 site.

In a second independent experiment, an alternative piece of partiallyhuman DNA 909 is used in place of the K-K DNA. The key features of thisDNA (referred to as “L-K”) (SEQ ID NO:3) are the following: a lox5171site; a neomycin resistance gene open reading frame lacking theinitiator methionine codon, but in-frame and contiguous with anuninterrupted open reading frame in the lox5171 site; a transcriptiontermination/polyadenylation sequence; an FRT site; an array of 38 humanlambda variable region gene segments, each comprised of human codingsequences embedded in mouse noncoding sequences; a 13.5 Kb piece ofgenomic DNA from immediately upstream of the cluster of J region genesegments in the mouse kappa chain locus; a 2 Kb piece of DNA containing5 human J lambda region gene segments embedded in mouse noncoding DNA; aloxP site in opposite relative orientation to the lox5171 site.

The transfected clones from the K-K (SEQ ID NO:2) and L-K (SEQ ID NO:3)transfection experiments are placed under G418 selection, which enrichesfor clones of cells that have undergone a recombinase-mediated cassetteexchange process in which the partially human donor DNA is integrated inits entirety into the deleted immunoglobulin kappa chain locus betweenthe loxP and lox5171 sites that were placed there by the 3′ vectors 905and 5′ vectors 903 respectively. The DNA region created using the K-Ksequence is illustrated at 911 in FIG. 9. The remaining elements fromthe 5′ vector 903 are removed via FLP-mediated recombination 906resulting in the final humanized locus as shown at 913.

G418-resistant ES cell clones are analyzed by PCR and Southern blot todetermine if they have undergone the expected recombinase-mediatedcassette exchange process without unwanted rearrangements or deletions.Both K-K and L-K clones that have the expected genomic structure areselected for further use.

The K-K ES cell clones and the L-K ES cell clones, each carrying thepartially human immunoglobulin DNA in the mouse kappa chain locus, aremicroinjected into mouse blastocysts from strain DBA/2 to createpartially ES cell-derived chimeric mice according to standardprocedures. Male chimeric mice with the highest levels of EScell-derived contribution to their coats are selected for mating tofemale mice. The female mice of choice for use in the mating are of theC57B1/6NTac strain, and will also carry a transgene encoding the Flprecombinase that is expressed in their germline. Offspring from thesematings are analyzed for the presence of the partially humanimmunoglobulin kappa chain locus, and for loss of the FRT-flankedneomycin resistance gene that was created in the recombinase-mediatedcassette exchange step. Mice that carry the partially human locus areused to establish colonies of K-K and L-K mice.

Mice carrying the partially human (i.e., humanized) heavy chain locus,produced as described in Example 3, can be bred with mice carrying ahumanized kappa chain locus. Their offspring are in turn bred togetherin a scheme that ultimately produces mice that are homozygous for bothhumanized loci, i.e., humanized for heavy chain and kappa. Such miceproduce partially human heavy chains comprised of human variable domainsand mouse constant domains. They also produce partially human kappaproteins comprised of human kappa variable domains and the mouse kappaconstant domain from their kappa loci. Monoclonal antibodies recoveredfrom these mice are comprised of human variable domains paired withhuman kappa variable domains.

A variation on the breeding scheme involves generating mice that arehomozygous for the humanized heavy chain locus, but heterozygous at thekappa locus such that on one chromosome they have the K-K humanizedlocus and on the other chromosome they have the L-K humanized locus.Such mice produce partially human heavy chains comprised of humanvariable domains and mouse constant domains. They also produce partiallyhuman kappa proteins comprised of human kappa variable domains and themouse kappa constant domain from one of their kappa loci. From the otherkappa locus, they will produce partially human lambda proteins comprisedof human lambda variable domains the mouse kappa constant domain.Monoclonal antibodies recovered from these mice are comprised of humanvariable domains paired in some cases with human kappa variable domainsand in other cases with human lambda variable domains.

Example 5 Introduction of a Partially Human Immunoglobulin Region intothe Immunoglobulin Lambda Chain Gene Locus of a Mouse Genome

A method for replacing a portion of a mammalian genome with partiallyhuman immunoglobulin region is illustrated in FIG. 10. This methodprovides deleting approximately 194 Kb of DNA from the wild-type mouseimmunoglobulin lambda locus 1001 by a homologous recombination processinvolving a targeting vector 1003 that shares identity with the locusboth upstream of the V2 gene segment and downstream of the V1 genesegment in the immediate vicinity of the J3, C3, J1 and C1 genesegments. The vector replaces the 194 Kb of DNA with elements designedto permit a subsequent site-specific recombination in which a non-nativepiece of DNA is moved into the modified V_(L) locus viarecombinase-mediated cassette exchange 1002. In this example, thenon-native DNA is a synthetic nucleic acid comprising both human andnon-human sequences.

The key features of the gene targeting vector 1003 for accomplishing the194 Kb deletion are as follows: a negative selection gene such as a geneencoding the A subunit of the diphtheria toxin or a herpes simplex virusthymidine kinase gene; 4 Kb of genomic DNA from 5′ of the mouse V2variable region gene segment in the lambda locus; an FRT site; a pieceof genomic DNA containing the mouse Polr2a gene promoter; a translationinitiation sequence (methionine codon embedded in a “Kozak” consensussequence); a mutated loxP recognition sequence (known as a lox5171 site)for the Cre recombinase; a transcription termination/polyadenylationsequence; an open reading frame encoding a protein that confersresistance to puromycin; this open reading frame would be on theantisense strand relative to the Polr2a promoter and the translationinitiation sequence next to it; it would also be followed by its owntranscription termination/polyadenylation sequence; a loxP recognitionsequence for the Cre recombinase; a translation initiation sequence (amethionine codon embedded in a “Kozak” consensus sequence) on the same,antisense strand as the puromycin resistance gene open reading frame; achicken beta actin promoter and cytomegalovirus early enhancer elementoriented such that it directs transcription of the puromycin resistanceopen reading frame, with translation initiating at the initiation codondownstream of the loxP site and continuing back through the loxP siteinto the puromycin open reading frame all on the antisense strandrelative to the Polr2a promoter and the translation initiation sequencenext to it; a mutated recognition site for the Flp recombinase known asan “F3” site; a 7.3 Kb of genomic DNA containing the J3, C3, J1 and C1gene segments and surrounding sequences; a second negative selectiongene such as a gene encoding the A subunit of the diphtheria toxin or aherpes simplex virus thymidine kinase gene.

Mouse embryonic stem (ES) cells (derived from C57B1/6NTac mice) aretransfected 1002 by electroporation with the targeting vector 1003according to widely used procedures. The resulting construct 1005 willreplace the native DNA with the sequences from the targeting vector 1003in the 196 Kb region.

Prior to electroporation, the vector DNA is linearized with arare-cutting restriction enzyme that cuts only in the prokaryoticplasmid sequence or the polylinker associated with it. The transfectedcells are plated and after ≧24 hours placed under drug selection usingpuromycin. Colonies of drug-resistant ES cells are physically extractedfrom their plates after they became visible to the naked eye over a weeklater. These picked colonies are disaggregated, re-plated in micro-wellplates, and cultured for several days. Thereafter, each of the clones ofcells are divided such that some of the cells are frozen as an archive,and the rest used for isolation of DNA for analytical purposes.

DNA from the ES cell clones is screened by PCR using a widely usedgene-targeting assay design. Four assays are used, and in each case oneof the PCR oligonucleotide primer sequences maps outside the region ofidentity shared between the targeting vector and the genomic DNA, whilethe other maps within the novel DNA between the two arms of genomicidentity in the vector (e.g., in the puro gene). According to thestandard design, these assays detect pieces of DNA that would only bepresent in clones of cells derived from transfected cells that hadundergone fully legitimate homologous recombination between thetargeting vector 1003 and the native DNA 1001.

Approximately six PCR-positive clones from the transfection 1002 areselected for expansion followed by further analysis using Southern blotassays. The Southern blots involve three probes and genomic DNA from theclones that has been digested with multiple restriction enzymes chosenso that the combination of probes and digests allow identification ofwhether the DNA has been properly modified by homologous recombination.

The six PCR-positive clones of ES cells are analyzed karyotypicallyusing an in situ fluorescence hybridization procedure designed todistinguish the most commonly arising chromosomal aberrations that arisein mouse ES cells. Clones that show evidence of aberrations will beexcluded from further use. Karyoptypically normal clones that are judgedto have the expected correct genomic structure based on the Southernblot data are selected for further use.

The ES cell clones carrying the deletion in one of the two homologouscopies of their immunoglobulin lambda chain locus are retransfected 1004with a Cre recombinase expression vector together with a piece of DNA1007 (SEQ ID NO:4) comprising a partially human immunoglobulin lambdachain locus containing V, J and C region gene segments. The key featuresof this piece of DNA 1007 are as follows: a lox5171 site; a neomycinresistance gene open reading frame (lacking the initiator methioninecodon, but in-frame and contiguous with an uninterrupted open readingframe in the lox5171 site); a transcription termination/polyadenylationsequence; an FRT site; an array of 38 human lambda variable region genesegments, each comprised of human lambda coding sequences embedded inmouse lambda noncoding sequences; an array of J-C units where each unitis comprised of a human J lambda region gene segment and a mouse lambdaconstant domain gene segment embedded within noncoding sequences fromthe mouse lambda locus (the human J region gene segments will be thoseencoding J1, J2, J6 and J7, while the mouse lambda constant domain genesegments will be C1 and/or C2 and/or C3); a mutated recognition site forthe Flp recombinase known as an “F3” site; an open reading frameconferring hygromycin resistance; the open reading frame is located onthe antisense strand relative to the immunoglobulin gene segment codinginformation in the construct; a loxP site in opposite relativeorientation to the lox5171 site.

The transfected clones are placed under G418 and/or hygromycinselection, which enriches for clones of cells that have undergone arecombinase-mediated cassette exchange process in which the partiallyhuman donor DNA is integrated in its entirety into the deletedimmunoglobulin lambda chain locus between the loxP and lox5171 sitesthat were placed there by the gene targeting vector. The remainingelements from the targeting vector 1003 are removed via FLP-mediatedrecombination 1006 resulting in the final humanized locus as shown at1011.

G418/hygromycin-resistant ES cell clones are analyzed by PCR andSouthern blot to determine if they have undergone the expectedrecombinase-mediated cassette exchange process without unwantedrearrangements or deletions. Clones that have the expected genomicstructure will be selected for further use.

The ES cell clones carrying the partially human immunoglobulin DNA 1011in the mouse lambda chain locus are microinjected into mouse blastocystsfrom strain DBA/2 to create partially ES cell-derived chimeric miceaccording to standard procedures. Male chimeric mice with the highestlevels of ES cell-derived contribution to their coats are selected formating to female mice. The female mice of choice here will be ofC57B1/6NTac strain, which carry a transgene encoding the Flp recombinaseexpressed in their germline. Offspring from these matings are analyzedfor the presence of the partially human immunoglobulin lambda chainlocus, and for loss of the FRT-flanked neomycin resistance gene and theF3-flanked hygromycin resistance gene that were created in therecombinase-mediated cassette exchange step. Mice that carry thepartially human locus are used to establish a colony of mice.

In some aspects, the mice comprising the humanized heavy chain and kappalocus (as described in Examples 3 and 4) are bred to mice that carry thehumanized lambda locus. Mice generated from this type of breeding schemeare homozygous for the humanized heavy chain locus, and can behomozygous for the K-K humanized locus or the L-K humanized locus.Alternatively, they can be heterozygous at the kappa locus carrying theK-K locus on one chromosome and the L-K locus on the other chromosome.Each of these mice will be homozygous for the humanized lambda locus.Monoclonal antibodies recovered from these mice will be comprised ofhuman variable domains paired in some cases with human kappa variabledomains and in other cases with human lambda variable domains. Thelambda variable domains will derive from either the humanized L-K locusor the humanized lambda locus.

Example 6 Introduction of a Partially Human Immunoglobulin Minigene intoa Mouse Genome

In certain other aspects, the partially human immunoglobulin region willcomprise a human variable domain minigene such as the one illustrated inFIG. 11. Here instead of a partially human immunoglobulin regioncomprising all or substantially all of the human V_(H) genes, the mouseimmunoglobulin region is replaced with a minigene 1119 comprising fewerhuman V_(H) genes, e.g. 1-43 human V_(H) genes.

A site-specific targeting vector 1129 comprising the partially humanimmunoglobulin region 1110 to be introduced to the mammalian host genomeis introduced 1102 to the genomic region 1101 with the deletedendogenous immunoglobulin region comprising the site-specificrecombination sites (1109, 1111, 1107 and 1105) and the puroΔTK gene1103. The site-specific targeting vector comprised a partially humanimmunoglobulin region comprising i) a V_(H) region 1119 comprising all44 human V_(H) coding regions and intervening sequences based on themouse genome endogenous sequences; ii) a 10 kb pre-DJ region 721comprising mouse sequence; iii) a DJ region 1125 comprising human D andJ coding regions and intervening sequences based on the mouse genomeendogenous sequences; and iv) a mouse non-functional J_(H) gene region.The partially human immunoglobulin region is flanked by recombinationsites 1109, 1111, 1105 and 1107) that will allow recombination with themodified endogenous locus. Upon introduction of the appropriaterecombinase 1104, the partially human immunoglobulin region isintegrated into the genome upstream of the constant gene region 1127.

As described in Example 1, the primary screening for introduction of thepartially human immunoglobulin variable region locus can be carried outby Southern blot, or with primary PCR screens supported by secondaryscreens with Southern and/or loss-of-native-allele qPCR screens. Thedeletion of the HPRT gene 1105 as part of the recombination event willallow identification of the cells that did not undergo the recombinationevent using (6-thioguanine-dependent) negative selection.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims. In the claims thatfollow, unless the term “means” is used, none of the features orelements recited therein should be construed as means-plus-functionlimitations pursuant to 35 U.S.C. §112, ¶6.

1-57. (canceled)
 58. A transgenic non-human vertebrate with a genomecomprising an introduced immunoglobulin region in a non-human vertebratehost, said introduced region comprising human immunoglobulin variableregion coding sequences and non-coding sequences based on an endogenousimmunoglobulin variable region locus of the non-human vertebrate host.59. The transgenic animal of claim 58, wherein the introducedimmunoglobulin region locus comprises human V_(H), D_(H), and J_(H),coding sequences, or V_(L) and J_(L) coding sequences, wherein L refersto Lambda or Kappa.
 60. The transgenic animal of claim 58, wherein thenon-human vertebrate host is a rodent.
 61. The transgenic animal ofclaim 60, wherein the non-human vertebrate host is a mouse.
 62. Thetransgenic non-human vertebrate of claim 58, wherein part or all of theendogenous variable genes in the immunoglobulin variable region locus ofthe non-human vertebrate host are removed.
 63. The transgenic animal ofclaim 62, wherein the introduced immunoglobulin region locus compriseshuman V_(H), D_(H), and J_(H), coding sequences, or V_(L) and J_(L)coding sequences, wherein L refers to Lambda or Kappa.
 64. Thetransgenic animal of claim 63, wherein the non-human vertebrate host isa rodent.
 65. The transgenic animal of claim 64, wherein the non-humanvertebrate host is a mouse.
 66. A method for generating the transgenicvertebrate of claim 58, said method comprising: introducing two or moretargeting sites for one or more site-specific recombinases into anon-human vertebrate cell and integrating at least one site in thecell's genome upstream and at least one site downstream of a genomicregion comprising the endogenous immunoglobulin variable gene segments,wherein the endogenous immunoglobulin variable gene segments comprise V,D and J gene segments, or V and J gene segments; providing a vectorcomprising a partially human immunoglobulin variable region having humancoding sequences and non-coding sequences based on an endogenousimmunoglobulin variable region locus flanked by site-specificrecombination sites, wherein the recombination sites are capable ofrecombining with those introduced into the vertebrate cell in step a);introducing the vector of step b) and a site-specific recombinasecapable of recognizing the recombinase sites in it; allowing arecombination event to occur between the genome of the cell and thepartially human immunoglobulin variable region locus, resulting in areplacement of the endogenous immunoglobulin variable region locus withthe partially human immunoglobulin variable region locus; selecting acell which comprises the partially human immunoglobulin region generatedin step d); and utilizing the cell to create a transgenic animalcomprising the partially human immunoglobulin region.
 67. The method ofclaim 66, wherein the cell is a mammalian embryonic stem (ES) cell. 68.The method of claim 66, further comprising the step of deleting theportion of the endogenous immunoglobulin region of the genome byintroduction of a recombinase that recognizes a first set ofsite-specific recombination sites, wherein such deletion in the genomeleaves in place a second set of site-specific recombination sites thatare not capable of recombining with one another after said introducingstep and before said providing step.
 69. The method of claim 68, whereinthe cell is a mammalian embryonic stem (ES) cell.